Name: Embryonic Heart Cell Thawing and Removal of Dead Cells
Uploaded: 2023-05-12T14:30:00
Description: Watch this Scientific Journal Video about Embryonic Heart Cell Thawing and Removal of Dead Cells at JoVE.com

embryonic heart cell thawing and removal of dead cells

Aislamiento de núcleos de células progenitoras cardíacas de ratón con resolución de una sola célula

Among birth defects, congenital heart defects (CHDs) are the most common, occurring in about 1% of live births each year1,2. Genetic mutations are identified in only a minority of cases, implying that other causes, such as abnormalities in gene regulation, are involved in the etiology of CHD2,3. Cardiac development is a complex process of diverse and interacting cell types, making the identification of causal noncoding mutations and their effects on gene regulation challenging. Organogenesis of the heart begins with cellular progenitors that give rise to different subtypes of cardiac cells, including myocardial, fibroblast, epicardial, and endocardial cells4,5.&#160;Single-cell genomics is emerging as a key method for studying heart development and assessing the impact of cellular heterogeneity in health and disease6. The development of multi-omics methods for the simultaneous measurement of different parameters and the expansion of computational pipelines has facilitated the discovery of cell types and subtypes in the normal and diseased heart6. This article describes a reliable single-nucleus isolation protocol for frozen cardiac progenitor cells obtained from mouse embryos that is compatible with downstream snRNA-seq and snATAC-seq (as well as snRNA-seq and snATAC-seq combined)7,8,9.
ATAC-seq is a robust method that allows the identification of regulatory open chromatin regions and the positioning of nucleosomes10,11. This information is used to draw conclusions about the location, identity, and activity of transcription factors. The activity of chromatin factors, including remodelers, as well as the transcriptional activity of RNA polymerase, can, thus, be analyzed since the method is highly sensitive for measuring quantitative changes in chromatin structure1,2. Thus, ATAC-seq provides a robust and impartial approach to uncovering the mechanisms controlling transcriptional regulation in a specific cell type. ATAC-seq protocols have also been validated to measure chromatin accessibility in single cells, revealing variability in the chromatin architecture within cell populations10,12,13.
Although there have been notable advances in the field of single cells in recent years, the main difficulty is the processing of the fresh samples needed to perform these experiments14. To circumvent this difficulty, various tests have been carried out with the aim of conducting analyses such as snRNA-seq and snATAC-seq with frozen cardiac tissue or cells15,16.
Several platforms have been used to analyze single-cell genomics data17. The widely used platforms for single-cell gene expression and ATAC profiling are platforms for multiple microfluidic droplet encapsulation17. As these platforms use microfluidic chambers, debris or aggregates can clog the system, resulting in non-usable data. Thus, the success of single-cell studies depends on the accurate isolation of individual cells/nuclei.
The protocol presented here uses a similar approach to recent studies using snRNA-seq and snATAC-seq to understand congenital heart defects18,19,20,21,22,23. This procedure utilizes the enzymatic dissociation of freshly microdissected cardiac tissue followed by the cryopreservation of mouse cardiac progenitor cells. After thawing, the viable cells are purified and processed for nuclear isolation. In this work, this protocol was successfully used to obtain snRNA-seq and snATAC-seq data from the same nuclear preparation of mouse cardiac progenitor cells.

Autor destacado: Aislamiento de núcleos de células progenitoras cardíacas de ratón para el perfil de epigenoma y expresión génica a resolución de una sola célula  

Aislamiento de núcleos de células progenitoras cardíacas de ratón para epigenoma y perfil de expresión génica a resolución de una sola célula

The main objective of this protocol is to study the interaction of genetic and environmental factors in the context of heart development and their contribution to congenital heart defects. Single nuclei approaches such as single nuclei RNA-seq and single nuclei ataxic are emerging as key methods to study cellular heterogeneity in health and disease during heart development. One limiting step of these experiments is that all the sample have to be run simultaneously.While working with all the experimental conditions, collecting fresh enough material is for all the condition simultaneously could be challenging. Although there have been a significant progress in the field in single cell for recent years, the main difficulty is processing free samples. Avoiding the difficulty is extremely benefit to identify the molecular mechanism underlying congenital heart disease defects.This protocol describes the steps to follow for successfully isolating high quality nuclei from frozen cardiac cell suspension obtained from mouse embryos. And our protocol is compatible with downstream single nuclei RNA-seq and single nuclei ataxic. We hope that this procedure will help interested researchers and encourage them to use this powerful method for their research.

Watch this Scientific Journal Video about Embryonic Heart Cell Thawing and Removal of Dead Cells at JoVE.com

Embryonic Heart Cell Thawing and Removal of Dead Cells  

Descongelación de células cardíacas embrionarias y eliminación de células muertas

Comience colocando la suspensión de células cardíacas embrionarias disociadas congeladas que contienen viales criogénicos en un baño de agua a 37 grados centígrados durante uno o dos minutos. Agregue un mililitro de medio completo, precalentado a 37 grados centígrados a la suspensión descongelada y mezcle tres veces con una pipeta. Luego transfiera la suspensión a un tubo cónico de 15 mililitros, que contenga 10 milímetros de medio completo caliente.Pase toda la suspensión celular sobre un colador de 30 micrómetros y enjuague el colador con uno o dos mililitros de medio caliente y completo. Recoja el flujo en el mismo tubo cónico. A continuación, centrifugar los tubos a 300 G durante cinco minutos.Después de retirar el sobrenadante, lave el pellet con PBS y transfiera la suspensión celular a un microtubo de 1,5 mililitros. Centrifugar la suspensión celular y añadir 100 microlitros de las microperlas magnéticas proporcionadas a las células granuladas. Homogeneizar la suspensión cinco veces utilizando una punta de pipeta de jabalí ancha antes de 15 minutos de incubación a temperatura ambiente.Mientras tanto, enjuague la columna de separación magnética con 500 microlitros de tampón de unión. Una vez completada la incubación, diluya la mezcla de suspensión de células de microperlas utilizando 500 microlitros de tampón de unión. A continuación, agregue 0.6 mililitros de la suspensión celular a la columna de separación magnética.Recoger el efluente de células vivas en un tubo de centrífuga de 15 mililitros. A continuación, enjuague el microtubo de 1,5 mililitros que contenía la suspensión celular con un mililitro de tampón de unión. Después del enjuague, transfiera el tampón de unión del microtubo de 1.5 mililitros a la columna y recoja el efluente en el mismo tubo de 15 mililitros.Repita el enjuague del tubo de 1,5 mililitros utilizando un mililitro de tampón de unión. Después de centrifugar el efluente a 300 G durante cinco minutos, retire el sobrenadante sin interrumpir el pellet celular. Añadir un mililitro de la solución BSA de PBS y mezclar suavemente pipeteando con una punta de pipeta de orificio ancho.Luego transfiera la suspensión celular a un microtubo de 1.5 mililitros. Una vez más, centrifugar la suspensión celular y retirar el sobrenadante sin interrumpir el pellet celular. Repita los dos lavados de un mililitro de PBS BSA.Después de retirar el mililitro de PBS BSA, resuspenda las células en 100 microlitros de solución de PBS BSA. Mezclar pipeteando 10 veces. Determinar la concentración mediante la realización de un ensayo de azul de tripano para asegurar un recuento de células de más o igual a 100.000 células y realizar una evaluación basada en la fluorescencia para la viabilidad de las células.El paso adicional de clasificar y eliminar las células muertas después de la descongelación permitió la obtención de una suspensión celular más limpia con una viabilidad celular significativamente mayor y mejoró la calidad de la muestra inicial. Para mayor precisión, se utilizaron dos métodos de conteo diferentes para cuantificar las células y los núcleos, incluido el azul de tripano y la evaluación de la viabilidad basada en la fluorescencia. En este protocolo, se observó que la viabilidad celular pasó del 80 al 90% antes del aislamiento de los núcleos a menos del 5% después del aislamiento de los núcleos.

Research

JoVE Journal

Developmental Biology

Embryonic Heart Cell Thawing and Removal of Dead Cells

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Investigación

Biología del Desarrollo

Mouse Cardiac Progenitor Cell Isolation, Digestion, and Freezing

Aislamiento, digestión y congelación de células progenitoras cardíacas de ratón

Nuclei Isolation from Cardiac Progenitor Cells

Aislamiento de núcleos de células progenitoras cardíacas